Method and apparatus for culturing micro-organisms



2 Sheets-Sheet 1 f//MLM July 6, 1965 c. F. KRABBE ETAL METHOD ANDAPPARATUS FOR CULTURING MICRO-ORGANISMS Filed Deo. 21, 1961 July 6, 1965METHOD AND APPARATUS FOR CULTURING MIGRO-ORGANISMS Filed Dec. 21, 1961 yc. F. KRABBE ETAL 3,193,460

2 Sheets-Sheet 2 (//XMLWMA A T roe/Veys,

United States Patent O s iss ses Marr-ron AND APrnArUs non CULGivneno-oaoANrsMs Carl lF. Krabbe, Council Blus, Iowa, and Frederick D.-

Taylor, Ralston, Nehr., assignors to Eli Lilly and Company,Indianapolis, ind., a corporation of Indiana Filed Dec. 21, 1961, Ser.No. 161,208 Claims. (Cl. 167-78) This invention relates to theproduction of Brucella and like organisms, as for use in preparingvaccines, and especially to an improved method vand apparatus for thispurpose.

In the production of Brucellosis vaccine, it is necessary to grow theBrucella organism on the surface of a growth medium, since growth inliquid medium, especially in the case of certain desirable strains ofBrucella organism, results in rapid dissociation of the organism and theproduction of a rough colony which has reduced antigenic propertiesinstead of the desired smooth colony which has high antigenicproperties.

In one common prior practice, the Brucella has been grown on the surfaceof solidied agar medium in culture bottles such as 5-liter Roux culturebottles. See for example Bulletin AR S-91-24, July 1960, BrucellaAbortus Strain 19 Vaccine, published by the Agricultural ResearchService of the United States Department of Agriculture. Quantityproduction by this method requires the handling of many bottles and theeventual pooling of theproduct from many bottles, which procedureinvolves many exposures of the product to contamination, and requiresmuch labor and expense.

It is the object of this invention to provide a method and apparatus forthe production of Brucella and other like organisms, as for vaccinepreparation, which greatly reduces thenumber of containers required tobe handled in the production of a given quantity of vaccine, whichfacilitates sterilization of the apparatus and growth rnedia and greatlyreduces the number and danger of exposures of the growth media tocontamination, which provides a. large area for surface growth of theorganism, which facilitates the preparation of the growthmedia and theinoculation thereof with the organism, and which provides for convenientand effective harvesting ofthe culture produced.

In accordance with the invention, the method of producing the organismcomprises depositing a layer of solid growth medium on the innercylindrical surface of a cylindrical container, or on an equivalentline-generated surface such as the conic surface of a truste-conicalcontainer. Conveniently, the medium used is one which is liquid at anelevated temperature and which solidies on cooling. Potato infusion agarmedium described in the above-mentioned Bulletin AR S-9l-24 is such amedium. With such a medium the growth layer is desirably prepared byintroducing a suitable quantity of the vmedium into the cylindricalcontainer, for example, sufficient to form a coating of the order or" lcm. thick on its cylindrical wall, sterilizing the whole with heat at atemperature which leaves the medium in liquid state, and then, desirablyafter partial cooling at room temperature, Flowing the still-liquidmedium over the inner surface of the cylindrical container wall, as byrotating the cylindrical container on its axis in a horizontal position,while cooling such wall to solidify the medium in a solid layer thereon.The container desirably has a smooth inner surface to facilitatecleaning, and the solidified layer is retained thereagainst andstabilized by a broken-surface support, such as an open mesh screenadjacent the container wall. The preparation of the growth medium mayalso include other steps, for example, steps of heating or storing underBQBAG@ Patented .July 6, 1965 ICC suitable conditions to ensure a propermoisture content before inoculation.

The cylindrical layer of growth-medium on the wall of the container isthen inoculated, or seeded, desirably by introducing a quantity ofliquid inoculum into the container and distributing the same over thegrowth medium. This may be done by again rotating the container for ashort time to flow the liquid inoculum over the entire inner surface ofthe solid cylindrical medium layer carried by the container wall. Theinoculated container is then incubated to cause the desired growth ofthe organism. For this, the containers may be stood on end so that anyexcess inoculum liquid drains to the bottom end. With Brucella, theinoculated growth medium may be incubated for a period .of about 72hours at a temperature of say 37 C. With Brucella and other organismswhich require aerobic conditions, and the container is arranged tocornmunicate with the atmosphere through a suitable lilter. Foranaerobic organisms, the incubation may be carried out with thecontainer sealed, or in the presence of inert or other suitable gases.

When the desired growth has occurred, the organism may be harvested byintroducing into the container a quantity of harvest liquid, such asBrucella stabilizer liquid, 4and again rotating the cylindricalcontainer on its axis in horizontal position to cause the liquid to washthe culture oil the growth surface. This produces a suspension ofculture organisms in the harvest liquid, and such suspension iswithdrawn for use, as by introducing a suction tube into the container.and withdrawing the liquid with suction.

The culture liquid obtained Iby this method corresponds to that obtainedin other culture methods, and may be processed in conventional ways forthe preparation of vaccine or for other conventional uses.

The method has been described with reference to a cylindrical container,since such containers are convenient and readily available and thereforepreferred, but the process can also be carried out in containers ofother lineegenerated shapes, such as frusto-conical containers, whichprovide inner surfaces generated by revolution of a straight line aboutan axis. Such a surface can be rotated about its axis of generation in aposition to carry the surface through a liquid supporting horizontalposition in which, as with cylindrical surfaces, a liquid will be evenlyllowed over the surface as the rotation occurs. For convenience, theterm line-generated surfaces is used to refer to such surfaces, and as aterm to include both cylindrical and conic surfaces.

The method described is especially adapted for the production ofBrucella abortus for the preparation of vaccine for vaccinating cattle.It may also be used for the production of other Brucella bacilli, suchas Brucella suis and Brucella melz'tensis; as well as for otherorganisms which are advantageously grown on the surface of solid stategrowth media, such as Hemophilus infiucnzae, Hemophlus pertussis,Pasteurellae species, Streptococceae species, Staphylococcus species,and Erysipetothrx rhusz'opathae.

In accordance with the apparatus aspects of the invention, thecylindrical container used is desirably a cylindrical stainless steeldrum provided with a removable drum head or lid having a breatheropening closed by an air filter and an access opening closed by aremovable closure. An open-mesh screen is fitted against the innercylindrical wall of the drum to facilitate formation and retention ofthe growth medium layer, and is desirably removable through the end ofthe drum when the lid is removed. A roller carriage is provided forrotating the drum on its axis in a horizontal position. This isconveniently a carriage having spaced rollers forming a cradle adaptedto support a drum in horizontal position and pro- 'enea/ieo vided with asuitable roller drive to rotate the supported Y drum on its axis. Thesame cradle may be used for a plurality of drums, and the drums removedtherefrom to simple stands or'rdollies during incubation. 1

The accompanying drawings illustrate the invention. nsuch drawings: n Y

FIG. 1 is an exploded view showing a processing container in accordancewith the invention, with the drum and certain other parts inlongitudinal section and with other parts in side elevation;

FiG. 2 is a longitudinal section on an enlarged'scale of the air iilterassembly shown in FG. 1;

. For convenience in sterilizing the container and theV growthmedia 60contained therein, the drum may be mounted on a wheeled dolly 62provided with suitable caster wheels 64, and the sterilization may becarried out in an autoclave 66 as shown in FIG. 3. Y

For the purpose of' distributing the growth media 60 over the innerVcylindrical Wall surface of the container 1t), the drum,y afterautoclaving, maybe mounted on a FlG. 3 is a diagrammatic view showingthe processing Y container in place in a treatment chamber, as forsterilizing under steam pressure or for incubating at controlledtemperature;

FiG. 4 is an end elevation of the processing container pacity and ofconventional construction. This includes a substantially cylindricallwall 12 having a pair of spaced circumferential beads 14 desirablyfitted with tire ringsV 16. One end wall of the drum is removable as alid 18 and may be secured in place by a ring clamp 20 and sealed with agasket 22. Y t

In accordance with the invention, the lid has two openings 24 andy 26,positioned at points spaced inward from the cylindrical wall of thedrum. Each opening is provided with a pipe iiange fitting 28, and theopening 24 is fitted with an inwardly extending tube for the receptionof an air filter 32. Such filter includes an outer end tube 34, and ismounted in sealed relation with the drum by means of a rubber stopper 36held in place by a pipe flange collar 3S. The opening 26 may be closedby a solid rubber stopper 4t) held in place by` a second flange collar3S. An open mesh cylindrical retention screen 42, made of stainlesssteel wire, is fitted inside the drum 1t), in resilient engagement withits cylindrical wall12.

T he air filter 32, shown on an enlarged scale in FIG. 2, l

consists of a cartridge having an outer cylindrical wall 44 closed atits outer end by anend Wall 46 connected to the outer breather tube 3d.vThe inner end of the cartridge is closedV by a cap 48 provided with acentral communication tube 50. The communication tubes 34 and 50 in theend walls of the cartridge 32 desirably project a short distance intothe cartridge and connect with open chambers 52 and 54 so that anyliquid which enters will be received and trapped in suchchambers'without clogging the openings. Between t'ne chambers 52 and 54the cartridge is subdivided by a plurality of transverse screens56, andthe space between such screens is filled with a fibrous filter material58 such as glass wool or cotton.

For use, the drum 16 shown in FIG. 1 and its associated parts areassembled in the relationship indicated. Lid 1S is placed on the end ofthe drum in sealing relationship with the gasket 22 and secured in placeby the clamping ring 2t). The air lter 32 is inserted in its tube`roller carriage 70 positioned over a drain 74, where it can be cooledwith Water as from a water spray head 72 or from a hose. The rollercarriage 70 conveniently consists of k a frame 73 mounted on wheelsV 75.Two shafts 76 rand 78 are mountedV in spaced parallel relation on theframe 7.3, and carry rollers 8th on which the drum 10 may besupported inhorizontal position. Conveniently the shaft 76 may be a fixed shaft andits rollers 80 maybe freely rotatable thereon, while the shaft 78 is arotatable shaft with some or all of its rollers St) fixed thereon, `andthe shaft 78 is connected through a gear reduction 82 to a motor 84 bywhich the shaft 73 and 'its rollers may be driven tov vrotate the drum10. in a f horizontal position onY its axis.

Y For withdrawing the harvest liquid from the drum 10, we may use theequipment shown in FIG. 6. This consists of a movable suction tube 90having a coarse inlet screen 92 at its free end, land connected by aflexible tube 94 to the inlet fitting 96 of a container 98 provided witha suction fitting 99 for connection to :a source of vacuum. Y Y Y Inusing the` apparatus shown rin the drawings for carrying out the newmethod of growing Brucella, the drum 10 is assembled as indicated inFIG. 1 and as described above to form a closed container whichcommunicates with the atmosphere only throughthe filter cartridge 32. Aquantity of a solid type culture medium for Brucella production is addedto each 55Y gallon drum in an amount sufficient to form a growth layerof desired thickness on the cylindrical call. This may -beA a thicknessof the order of from 0.5 to 1.5 cm. The drums vcontaining the culturemedium 64) are sterilized as in vautoclave 66 either on dollys asindicated in FIG.. 3 or in verticalposition. At sterilizingtemperaturerthe growth medium isrnorrnrally iny liquid state, and liesin a pool at the bottom of the drum 10. After sterilization, the drum isremoved from the autoclave and isV desirably Vpartially cooled lat roomtemperature, to a temperature -wall 12 is cooled by a spray of coolingwater from the spray-head 72 or by manual application of a stream o'fwater from a hose until the drum temperature is well below thesolidiication temperature of the medium.l This causes the culture mediumto solidify on the Wall 12 to f form a substantially uniform layer 61 onthe inner surface 3i), and the opening 24 is sealed by the rubberstopper 36V Y held in place by the pipe cap 38, with the breather tube34 extending through the stopper 36 and the cap 38. A quantity of growthmedium is introduced through the access opening 26 and such opening isclosed by the rubber stopper 4t) held in place by the pipe cap 38. Thecylinrical drum forms a sealed container which communicates with theatmosphere oniy through the Vcommunication tubes 34 and 5@ and thepacking 53 of the iilter 32.'.

of such wall 12. This is retained by the wire mesh 42 and issufficientlyr stable to permit reasonable handling of the drum 10,

When the solid layer 61 of culture medium has been formed onthe innersurface of the drum 10, a suitable quantity of liquid inoculum is addedto the drum through the access opening 26 in an amount suiiicient to beowed over the entire surface of the growth layer. This may be of theorder of 200 ml. of such liquid. The drum is then again rotated on itsaxis on the roll-er carriage 70 Y,to distribute the inoculum over theentire surface of the ycylindrical layer 61 of culture medium. Theinoculated drum may now be removed from the roller carriage 7tto thedolly 62, and placed on end in an incubation room where it is held at asuitable incubating temperature for a suilicient time to produce thedesired growth of the culture organism. In the case of Brucella, thismay be the order of 72 hours at 37 C.

At the end of the incubation period, the drum is removed from theincubation room and placed again on the roller dolly 70. A suitablequantity of harvest or wash liquid is then introduced into the drum,'for example approximately 2000 ml. of Brucella harvest liquid, and thedrum is rotated on its axis on the carriage 70 for a period of say fiveto ten minutes to cause the harvest liquid to gently flow over and washthe surface of the solid layer 61 of culture medium. This washes theculture cells olf the culture medium and places them in suspension inthe harvest liquid. The resulting liquid suspension is then withdrawnfrom the drum in any suitable manner. With the appartus shown in FIG. 6,such withdrawal is carried out by inserting the suction tube 90 throughthe access opening 26 of the drum into the pool of liquid in the drum10. By applying suction to the container 93 through the suctionconnection 99, the liquid will be withdrawn from the drum into thecontainer 98.

The treatment of the culture yorganisms produced forms no part of mypresent invention, and may be in accordance with the conventionalprocedure.

The invention is further illustrated by the following examples:

Example 1 Sixteen agar slants in 10U-ml. tubes, each tube containing 40ml. of solidified potato infusion agar (p. 7 of ARS-91-24), wereinoculated with Brucella abortus and incubated for approximately 72hours at 37 C. The resulting growth was washed from each tube with 10ml. of sterile buffered saline and transferred aseptically into a5-liter toxin bottle containing 450 ml. of solidified potato infusionagar. Sixteen bottles were thus inoculated and incubated at 37 C. for 72hours.

Into a clean stainless-steel cooker were charged 140 liters ofpotato-infusion agar, and the cooker was heated sufficiently to melt themedium. The melted material was dispensed into sixteen stainless-steeldrums 10 of the design described above, 8 liters per drum, and intotwenty-one 5-1iter toxin bottles, 450 ml. per bottle. The drums andbottles were steam-sterilized in an autoclave at 121 C. and 15 p.s.i.pressure for 45 minutes, and were removed from the autoclave after thetemperature had dropped below 100 C.

The bottles were cooled to room temperature in the horizontal position,the agar being allowed to gel on the deep side of the bottle. Thebottles were reserved for seed preparation in subsequent batches.

The drums were placed on the roller carriage 70 and, beginning to 20minutes after removal from the autoclave, were rolled on the carriage inhorizontal position and were cooled with a stream of water to roomtemperature, so that the medium solidified as a layer covering the innercylindrical surface of the drum.

Into each of the incubated seed bottles were aseptically introduced 200ml. of buffered saline and the bottle was agitated to wash the growthfrom the agar. The resulting suspension was examined microscopically byuse of Gram stain to detect any unsatisfactory cultures. The suspensionfrom each bottle was then transferred aseptically to a drum, after whichthe drum was rolled for 3 to 5 minutes, then incubated at rest inupright position for 72 hours at 37 C.

The incubated drums were examined for contamination, and one wasdiscarded. Into each of the remaining 15 drums were aseptically addedapproximately 2 liters of buffered saline solution `as Brucellastabilizer and harvest liquid (ARS-91-24), the drum was rolled forapproximately 10 minutes, and the resulting suspension was asepticallysyphoned into a S-liter toxin bottle. Each of the bottles was tested forbulk purity by inoculating 0.5

6. and 1.0 ml. samples into two tubes each of dextrosestarch agar anduid thioglycollate medium (total of 4 tubes), incubating at 37 C. for 72hours, and examining for contamination. One bottle was discarded.

The remaining 14 bottles were pooled and commingled with sterile skimmilk as a stabilizer. The cell concentration was measured, and thesuspension was metered into vaccine vials, frozen, and lyophilized.

Example 2 Example l is repeated, save that instead of using Brucellaabortus as the organism, Brucella suis is used. In this case theharvested suspension of Brunella suis organisms is processed inconventional manner to prepare a hog Vaccine.

As noted above, the invention can also be used for the production ofother organisms which are produced by surface growth on a stable solidgrowth medium.

We claim as our invention:

1. The method of producing -a culture of a surfacegrowth microorganism,which comprises (a) placing in a cylindrical container a quantity ofnormally solid culture medium which liquees under heat,

(b) sterilizing the container and culture medium at a temperature whichleaves the medium in liquid state,

(c) rotating the container on its axis in a horizontal position to flowthe liquid medium over the cylindrical wall of the container,

(d) meanwhile cooling said wall to solidify the culture medium on saidwall to form a surface-growth layer on the inner surface thereof,

(e) introducing a quantity of liquid inoculum and rotating the containerto distribute the inoculum over the surface-growth layer to inoculatethe same,

(f) subjecting the inoculated layer to incubation conditions to produceculture growth thereon,

(g) introducing a quantity of harvest liquid and rotating the containeron its axis to wash the harvest liquid over the growth-containingsurface to form a suspension of culture organisms in the harvest liquid,

(h) and recovering said suspension.

2. The method dened in claim 1,

(a) in which the container is incubated in upright position to drain anyexcess inoculum liquid to one end of the container.

3. The method defined in claim 1,

(a) with the addition of providing retention means adjacent said innerwall surface and solidifying the surface-growth layer in supportedrelation therewith.

4. The method defined in claim 1 in which the surfacegrowthmicroorganism is a Brucella organism.

5. The method defined in claim 1 in which the surfacegrowthmicroorganism is Brucella abortus.

6. The method of producing a culture of a surfacegrowth microorganism,which comprises (a) forming a retained solid cylindrical layer ofsurface-growth culture medium against the inner cylindrical surface of acylindrical container,

(b) distributing a quantity of liquid inoculum over the surface of saidlayer by rotating the container on its axis in a horizontal position,

(c) subjecting the inoculated retained layer to incubating conditions toproduce culture growth thereon,

(d) flowing a quantity of harvest liquid over the growthcontaining layerby rotating the container on its axis in a horizontal position tothereby produce a suspension of growth organisms in the harvest liquid,

(e) and recovering such harvest liquid.

7. The method of producing a culture of a surfacegrowth microorganism,which comprises (a) forming on the inner surface of a container wall ofline-generated configuration a layer of surface? (d) introducingaquantity of harvestrliquid and again` rotating said wall on its axisYin a position to flow the harvest liquid over the growth-containingsurface to form a suspension of culture organisms in the harvest liquid,and

(e) recovering said suspension. Y

8. The method of producing a culture of a surfacegrowthV microorganism,which comprises y (a) placing a quantityv of heat-liquefiable,normallysolid growth medium in a container having .a side wall ofline-generatedv configuration,

(b) rotating the container, with the growth medium lin liquidV statetherein, on its axis in a position to distribute the liquid overtheinner surface of said side wall, y

(c) cooling the side wall during suchrrotation to solidify the mediumand forma surface-growth layer on said inner surface, Y

(d) inoculating the surface of said layer,

(e) subjecting the inoculated layer to incubation con- (b) an open-meshliner removably mounted against the inner cylindrical Wall of the drum,

(c) an access port in an end wall of said drum and a closure for saidport,

(d) a lter fitting forming a breather opening in an end wall of thedrum, f

(e) an air filter for said breather opening, and l V(f) means to rotatethe drum on its axis in a horizontal position'for distributing aquantity of solidiable liquid over the yinner Vcylindrical surfacethereof, (g) said port and fitting being spaced inward from thecylindrical wall to avoid contact with the liquid during suchdistribution. Y 10.*Apparatus for producing a culture of a surface- Ygrowth microorganismi, comprising (a) a container having a continuouswall of linegenerated configuration and having an end opening at one endof said wall, Y Y Y (b) a removable end wall forming a closure -for saidopening and means for securing and sealing thesame in closing relationon` said container,

(c) retention means overlying the inner surface of saidv line-generatedrwall for retaining a layer of solidifiedgrowth medium thereon,

(d) said retention'means *being removable through Y said opening'whensaidfclosure is released from the container, y Y y i (e) and bearinglmeans Vforv supporting said container for rotation on` the axis of saidline-generated Wall in a position to flow soliditiable liquid over theinner surface of said Wall, 1

(f) and breather meansl for said container communicating with theinterior thereof at a point spaced inward from said innerV surface toavoid contact with liquid being flowed over said surface.

References Cited by the Examiner UNITED STATES PATENTS 2,686,754 8/54Monod 195-142 2,996,426 8/61 Toulmin 167--78 I FOREIGN PATENTS 625,1208/61 Canada. 850,611 10/60 Y Great Britain.

JULIAN S. LEVITT, Primary Examiner.

M. O. WVOLK, LEWIS GOTTS, Examiners.

1. THE METHOD OF PRODUCING A CULTURE OF A SURFACEGROWTH MICROORGANISM,WHICH COMPRISES (A) PLACING IN A CYLINDIRCAL CONTAINER A QUANTITY OFNORMALLY SOLID CULTURE MEDIUM WHICH LIQUEFIES UNDER HEAT, (B)STERILIZING THE CONTAINER AND CULTURE MEDIUM AT A TEMPERATURE WHICHLEAVES THE MEDIUM IN LIQUID STATE, (C) ROTATING THE CONTAINER ON ITSAXIS IN A HORIZONTAL POSITION TO FLOW THE LIQUID MEDIUM OVER THECYLINDRICAL WALL OF THE CONTAINER, (D) MEANWHILE COOLING SAID WALL TOSOLIDIFY THE CULTURE MEDIUM ON SAID WALL TO FORM A SURFACE-GROWTH LAYERON THE INNER SURFACE THEREOF, (E) INTRODUCING A QUANTITY OF LIQUIDINOCULUM AND ROTATING THE CONTAINER TO DISTRIBUTE THE INOCULUM OVER THESURFACE-GROWTH LAYER TO INOCULATE THE SAME, (F) SUBJECTING THEINOCULATED LAYER TO INCUBATION CONDITIONS TO PRODUCE CULTURE GROWTHTHEREON, (G) INTRODUCING A QUANTITY OF HARVEST LIQUID AND ROTATING THECONTAINER ON ITS AXIS TO WASH THE HARVEST LIQUID OVER THEGROWTH-CONTAINING SURFACE TO FORM A SUSPENSION OF CULTURE ORGANISMS INTHE HARVEST LIQUID, (H) AND RECOVERING SAID SUSPENSION.